(W1130-07-42) Inhaled Delivery of Synergistic Alginate Lyase-Ceftazidime Combination to Eradicate Pulmonary Pseudomonas aeruginosa Biofilms

Purpose: Pseudomonas aeruginosa (PA) forms biofilms that render antibiotic treatments ineffective and cause chronic lung infections [1]. Alginate lyases (AL), enzymes that degrade alginate of the PA biofilm matrix, have demonstrated promise in dispersing biofilms and enhancing the efficacy of antipseudomonal antibiotics, like ceftazidime (CFZ) [2]. Effective combination therapy using AL and CFZ against PA biofilms in the lungs of patients requires both drugs to reach the site of infection in sufficient concentrations and at a feasible synergistic ratio. This is likely to be benefitted from co-delivery of these agents directly to the lungs via inhalation, ensuring consistent lung deposition and maximizing local concentrations while minimizing systemic side effects [3]. However, an inhaled delivery approach suitable for the AL-CFZ combination has not yet been explored. This study aims to investigate the feasibility of applying AL to improve CFZ efficacy against pulmonary PA biofilms and delivering this combination via nebulization for inhaled therapy.

Methods: The combined effects of AL with CFZ on PAO1 bacterial growth and biofilm formation were evaluated using the microtiter plate assay. Next, the synergy of the AL-CFZ combination against pre-formed PAO1 biofilms was evaluated by measuring biofilm viability using the MBEC assay® kit. To elucidate the underlying synergistic mechanisms, confocal laser scanning microscopy (CLSM) imaging, Transwell biofilm penetration study and scanning electronic microscopy (SEM) imaging were performed. Finally, the aerosolization stability and deposition profiles of AL-CFZ nebulized by the commercialized Aeroneb® Go vibrating mesh nebulizer were examined.

Results: Although AL alone had no bactericidal effect, it enhanced CFZ activity against bacterial growth, biofilm formation and established biofilm eradication, resulting in a synergistic antibiofilm effect (Figure 1). Mechanistic studies showed that AL removed the biofilm exopolysaccharide layer surrounding bacteria, allowing CFZ diffusion throughout the biofilms efficiently to access bacterial cells and thus improving the biofilm eradication compared with CFZ-alone treatment (Figure 2). Nebulized by an Aeroneb® Go vibrating mesh nebulizer, the AL-CFZ combination exhibited comparable deposition patterns, with approximately 50% of the drug-containing aerosols being inhalable and likely depositing in the lower airways. In addition, this device caused negligible damage to both drugs, with the antibiofilm activity of the post-nebulized AL-CFZ combination was effectively retained (Figure 3). Collectively, the combination of AL and CFZ showed synergistic effects against PA biofilms, and their inhalation delivery using the Aeroneb® Go vibrating mesh nebulizer enabled efficient lung deposition with similar profiles for both agents and negligible loss of their antibiofilm activity.

Conclusion: This study highlighted the promise of inhalation delivery of synergistic combinations of biofilm-dispersing enzymes and antibiotics to treat chronic lung infections associated with biofilms.

References: 1. Parkins MD, Somayaji R, Waters VJ. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev. 2018;31(4):e00019-18. doi: 10.1128/CMR.00019-18.
2. Lamppa JW, Griswold KE. Alginate lyase exhibits catalysis-independent biofilm dispersion and antibiotic synergy. Antimicrob Agents Chemother. 2013;57(1):137-45. doi: 10.1128/AAC.01789-12.
3. Banat H, Ambrus R, Csoka I. Drug combinations for inhalation: Current products and future development addressing disease control and patient compliance. Int J Pharm. 2023; 643:123070. doi: 10.1016/j.ijpharm.2023.123070.

Acknowledgements: This work was supported by the University Grants Committee of Hong Kong (ref. 14112921) and the Guangdong-Hong Kong-Macao Joint Laboratory for New Drug Screening, School of Pharmacy, The Chinese University of Hong Kong. The support of HKPFS from the University Grants Committee to Honglan Wang was greatly acknowledged.


Figure 1. Percentage of bacterial growth (A, C) and biofilm biomass (B, D) after treatment with various concentrations of AL or CFZ for 24 h. (E) Percentage of biofilm biomass after treatment with various concentrations of AL in combination with 1 μg/mL of CFZ for 24 h. (F) Disk diffusion test of AL-CFZ combination against PAO1 bacteria on the LB agar plate and measurement of diameters of inhibition zones. (G) Percentage of biofilm viability after treatment with various concentrations of CFZ using resazurin as a detection dye. Inset indicated the purple-to-pink color change of resazurin after reacting with biofilms with varying viability in the wells. (H) Checkerboard synergy assay of AL-CFZ combination in eradicating established biofilms. (I) Percentage of biofilm viability after treatment with various concentrations of AL in combination with 512 μg/mL of CFZ.


Figure 2. CLSM observation of the penetration behavior of Sulfo-Cy3-labeled CFZ (red) within the biofilm matrix consisting of EPS stained by Alexa Fluro 488 labeled concanavalin A (green) after various treatments. PAO1 bacterial nuclei were stained by Hoechst 33342 (blue). 3D (A) and z-stack (B) images were visualized by Imaris software. Scale bar: 20 μm. Semi-quantification of the EPS thickness (C), Sulfo-Cy3-CFZ depth (D), and percentage of penetrated EPS thickness (E) at eight designated positions (indicated by red arrows) using Image J. (F) Schematic diagram of the transwell biofilm penetration assay. (G) Percentage of penetrated CFZ across PBS and AL-treated biofilms at 15- and 30-min. (H) SEM images of biofilm structural changes after various treatments. Scale bar: 5 μm.


Figure 3. Schematic diagram of the (A) Aeroneb® Go vibrating mesh nebulizer, (B) influence of particle size in lung deposition sites, and (C) MSLI experimental device. (D) Deposition profiles of AL and CFZ aerosols using the MSLI. The deposition fraction was calculated as the mass of drugs at each stage relative to the emitted dose. (E) Biofilm inhibition activity of AL-CFZ combination before or after nebulization.